SUPREM: an engineered non-site-specific m6A RNA methyltransferase with highly improved efficiency

N 6-Methyladenine (m6A) RNA methylation plays a key role in RNA processing and translational regulation, influencing both normal physiological and pathological processes. Yet, current techniques for studying RNA methylation struggle to isolate the effects of individual m6A modifications. Engineering of RNA methyltransferases (RNA MTases) could enable development of improved synthetic biology tools to manipulate RNA methylation, but it is challenging due to limited understanding of structure-function relationships in RNA MTases. Herein, using ancestral sequence reconstruction, we explore the sequence space of the bacterial DNA methyltransferase EcoGII (M.EcoGII), a promising target for protein engineering due to its lack of sequence specificity and its residual activity on RNA. We thereby created an efficient non-specific RNA MTase termed SUPer RNA EcoGII Methyltransferase (SUPREM), which exhibits 8-fold higher expression levels, 7°C higher thermostability and 12-fold greater m6A RNA methylation activity compared with M.EcoGII. Immunofluorescent staining and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed SUPREM's higher RNA methylation activity compared with M.EcoGII in mammalian cells. Additionally, Nanopore direct RNA sequencing highlighted that SUPREM is capable of methylating a larger number of RNA methylation sites than M.EcoGII. Through phylogenetic and mutational analysis, we identified a critical residue for the enhanced RNA methylation activity of SUPREM. Collectively, our findings indicate that SUPREM holds promise as a versatile tool for in vivo RNA methylation and labeling.